Column chromatography is a separation and/or purification technique in which a stationary “bed” of a packing medium is contained within a rigid tube. The packing medium can be in the form of particles of a solid (“stationary phase”) or a solid support material coated with a liquid stationary phase. Either way, the packing medium typically fills the inside volume of the column tube.
In separation chromatography, as a liquid sample (“mobile phase”) passes through the column, different compounds in the sample can associate differentially with the stationary phase such that they are slowed relative to the mobile phase and move through the column at different speeds. Thus, those compounds that associate more with the stationary phase move more slowly through the column than those that associate less, and this speed differential results in the compounds being separated from one another as they pass through and exit the column. Features of the stationary phase that promote differential association can be ionic charge (ion exchange chromatography), hydrophobicity (hydrophobic interaction chromatography), and porosity (size exclusion chromatography).
In yet another type of column chromatography, affinity chromatography, the packing medium includes binding agents, such as antigens, antibodies, or ligands, that specifically bind to one or more desired compounds or molecules in the liquid sample. Thus, as the liquid sample flows through the packing medium only the desired compounds or molecules remain in the column. A subsequent flow through the packing medium of an eluting liquid separates the desired compounds or molecules from the binding agents attached to the packing medium, or separates the binding agents from the packing medium. Either way, the desired compounds or molecules are rinsed out of the column and collected in the eluting fluid. Affinity chromatography can be used in a number of applications, including nucleic acid purification, protein purification from cell free extracts, and purification from blood.
The main components of a chromatography column are the tube, which is often made of a metal, glass, or highly rigid plastic material, and a pair of flow distributors, which are typically inserted into the two ends of the tube to form a space or chamber in the tube between the flow distributors into which the packing medium is loaded. In such columns a small space or void can form between the outer edge of the flow distributors and the inner wall of the column tube up to the point at which the O-ring is creating a seal between the flow distributors and the inner wall of the column tube. This void creates a so-called “dead zone” or “dead space” into which fluids and contaminants can enter and become entrapped and stagnant, rather than flowing through the medium within the column tube. In addition, such dead zones can become contaminated and are difficult to clean when the columns are to be reused.